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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-06-19 to 1997-08-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A well conducted study according to guidelines and done in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
No sampling was performed after the 48 hour time period.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 22-30 grams
- Assigned to test groups randomly: yes
- Housing: polypropylene cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 54-79
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Arachis oil BP
Frequency of treatment:
Single dose
Post exposure period:
24, 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250, 500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 males, 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide dosed at 50 mg/kg orally.

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range-finding study
TREATMENT AND SAMPLING TIMES : One group of mice from each dose level was sacrificed by cervical dislocation 24 hours following treatment and a second group, dosed at 500mg/kg, were sacrificed at 48 hours. Immediately following sacrifice (i.e. 24 or 48 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of rnicronucleated cells per 1000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values.

Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group. A positive mutagenic response was demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour samples when compared to their corresponding control group. If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test. A positive response for bone marrow toxicity was considered demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
The data was analysed following a √(x + 1)transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs, mortality
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Animals dosed by the oral route showed no clinical signs nor were any deaths observed.
In animals dosed via the intraperitoneal route, premature deaths occurred at and above 750 mg/kg. Clinical signs, hunched posture, ptosis, lethargy, ataxia, splayed gait and decreased respiratory rate were observed in animals dosed with 500 mg/kg.
Based on these results the intraperitoneal route of administration was selected for the main study. The maximum tolerated dose (MTD) was 500 mg/kg; this was therefore selected for the definitive study, in addition to 250 mg/kg and 125 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
In the 48 hour 500mg/kg dose group, three premature deaths were occurred. Clinical signs were observed in the 500mg/kg 24 and 48-hour dose groups, these included hunched posture, ptosis, lethargy, ataxia and splayed gait. The test material proved to be more toxic that the range finding study suggested, however the low number of premature deaths was not considered to adversely affect the study.
There were no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes (PCEs) in any of the test material dose groups when compared to their concurrent vehicle controls.

There were no decreases in the PCE/NCE (Normochromatic erythrocytes) ratio in the 24 or 48-hour treatment groups when compared to the concurrent control. The observation of the clinical signs and premature deaths in the 500 mg/kg dose group was taken to indicate that systemic absorption had occurred.
The positive control group showed a significant increase in the incidence of micronucleated PCEs therefore demonstrating the sensitivity of the system.
The results are summarised in Table 2 and presented individually in Table 3 attached below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
2,2,6,6-Tetramethyl-4-oxopiperidinooxy did not produce a significant increase in micronuclei in polychromatic erythrocyes of mice following an
intraperitoneal dose of 125, 250 or 500 mg/kg. The test material was not genotoxic.
Executive summary:

A GLP study was performed in accordance with OECD guideline 474 in order to assess the potential of 2,2,6,6,-tetramethyl-4-oxopiperdinooxy to produce damage to chromosomes or aneuploidy when administered to mice.

A range-finding study was performed to identify suitable dose levels and route of administration.

The micronucleus study was conducted via the intraperitoneal route in groups of 10 mice (five male/ five female) at the maximum tolerated dose (MTD) 500 mg/kg and 250 and 125 mg/kg at the lower doses. The test animals were sacrificed 24 or 48-hours after treatment, the bone marrow extracted and smear preparations made and stained. Polychromatic and monochromatic erythrocytes (PCE and NCE) were scored for the presence of micronuclei. Further groups of mice were treated with a single oral dose of arachis oil or cylclophosphamide to serve as vehicle and positive controls respectively.

The results showed no significant increase in the incidence of micronucleated PCE in animals dosed with the test material when compared to the concurrent vehicle controls. No statistically significant decrease was observed in the PCE/NCE ratio in either the 24 or 48-hour treatment groups when compared to the control. The observation of clinical signs in the 500 mg/kg dose group and the premature deaths observed in the 500 mg/kg 48 hour dose group were taken to indicate that systemic absorption had occurred.

The positive control produced a marked increase in the frequency of micronucleated PCEs demonstrating the sensitivity of the test system.

Under the conditions of the test, 2,2,6,6-tetramethyl-4-oxpiperidinooxy was considered to be non- gentoxic.